Methods and systems for pretreatment and processing of biomass

ABSTRACT

According to one embodiment of the invention, a system for processing biomass includes a water-impermeable bottom liner, a gravel layer supported by the bottom liner, a drain pipe disposed within the gravel layer, a biomass input device operable to deliver biomass over the gravel layer to form a biomass pile, a lime input device operable to deliver lime to the biomass for pretreating the biomass, a distribution pipe elevated above the gravel layer, and a pump operable to circulate water through the biomass pile by delivering water to the distribution pipe and receiving water from the drain pipe after it has traveled through the biomass pile.  
     According to another embodiment, a method for biomass pretreatment with alkali, conducted at ambient pressure for approximately 4-16 weeks at temperatures ranging from approximately 25° C. to 95° C. Biomass may be lignocellulosic biomass and may be rendered suitable for enzymatic digestion or pulp production.

PRIORITY CLAIM

[0001] The present application claims priority under 35 U.S.C. §119(e)to U.S. Provisional Patent Application Ser. No. 60/423,288 filed Nov. 1,2002.

STATEMENT OF GOVERNMENT INTEREST

[0002] Funding from the U.S. Department of Agriculture was used in thedevelopment of certain aspects of the present invention. Accordingly,the U.S. government may have certain rights therein.

TECHNICAL FIELD OF THE INVENTION

[0003] The present invention relates to processes for biomass treatment,including pretreatment. It also relates to apparatuses for the storage,pretreatment and enzymatic digestion, such as fermentation of suchbiomass.

BACKGROUND OF THE INVENTION

[0004] Treatment of biomass, especially waste biomass, to recover usefulsubstances has been the focus of numerous efforts. Such treatments haveused a variety of treatment methods and chemicals, depending upon thedesired recovery substance. Treatment with lime (Ca(OH)₂ or calciumhydroxide) has been attempted, but usually at temperatures above 60° C.for time frames of only a few weeks to a month.

[0005] High-temperature lime treatments have been used to enhanceenzymatic digestibility of biomass. One such process uses hot lime onlyand another uses hot lime+high-pressure oxygen.

[0006] Biomass processing is also useful in making pulp. The most commonmethods for making pulp for paper or cardboard are Kraft and sodapulping. Both of these methods use expensive chemicals and expensivetreatment vessels.

[0007] Additionally, previous methodologies and treatment systems haveoften required movement of the biomass several times during the entiretreatment process, including pretreatment and recovery. Aspects of thepresent invention may be used to overcome some of these and otherproblems associated with previous methodologies.

SUMMARY OF THE INVENTION

[0008] One embodiment of the invention relates to a system forprocessing biomass, including:

[0009] a water-impermeable bottom liner;

[0010] a gravel layer supported by the bottom liner;

[0011] a drain pipe disposed within the gravel layer;

[0012] a biomass input device operable to deliver biomass over thegravel layer to form a biomass pile;

[0013] a lime input device operable to deliver lime to the biomass forpretreating the biomass;

[0014] a distribution pipe elevated above the gravel layer; and

[0015] a pump operable to circulate water through the biomass pile bydelivering water to the distribution pipe and receiving water from thedrain pipe after it has traveled through the biomass pile.

[0016] In more specific embodiments, the biomass may be lignocellulosicbiomass, such as bagasse and corn stover. The gravel layer may beapproximately three feet thick. The lime input device may be operable todeliver lime to the biomass either during or after the delivering of thebiomass over the gravel layer. Lime may be delivered to the biomass inan amount between approximately 10% and 30% of the biomass by weight.Lime pretreatment may occur a temperature between approximately 25° C.and 95° C. at ambient pressure and for a time period greater thanapproximately four weeks.

[0017] The system may also include a heat exchanger coupled to thedistribution pipe and operable to control a temperature of the waterthat is delivered to the distribution pipe. It may also include an airblower and an air distribution pipe operable to deliver air to thebiomass pile. A container of lime water slurry may coupled to the airdistribution pipe and operable to scrub the air of carbon dioxide beforethe air is delivered to the biomass pile. A a calcium carbonate inputdevice may be added to deliver calcium carbonate to the biomass forpretreating the biomass.

[0018] The system may also include an inoculum input device operable todeliver an inoculum to the biomass pile for fermentation of the biomasspile.

[0019] Another embodiment of the present invention relates to a systemfor processing biomass, including:

[0020] a water-impermeable bottom liner;

[0021] a grid-like lattice structure coupled to the bottom liner to forma roof;

[0022] a geomembrane coupled to the grid-like lattice structure;

[0023] a gravel layer supported by the bottom liner;

[0024] a plurality of drain pipes disposed within the gravel layer;

[0025] a conveyor belt coupled to the top liner and operable to deliverbiomass over the gravel layer to form a biomass pile;

[0026] a lime input device operable to deliver lime to the biomass forpretreating the biomass;

[0027] a plurality of distribution pipes coupled to the top liner andassociated with respective ones of the plurality of drain pipes; and

[0028] a plurality of pumps coupled to respective ones of the pluralityof drain pipes and respective ones of the plurality of distributionpipes, the pumps operable to circulate water through the biomass pile bydelivering water to the distribution pipes and receiving water from thedrain pipes after the water has traveled through the biomass pile.

[0029] In more specific embodiments, the biomass may be lignocellulosicbiomass such as bagasse and corn stover. The grid-like lattice structuremay be formed from a plurality of I-beams in a general shape of a halfcylinder. A foam layer may be coupled to an outside of the geomembrane.

[0030] The system may also include a sugar extraction device operable toextract sugar from a raw feedstock to produce the biomass. The rawfeedstock may be energy cane or sweet sorghum. The sugar extractiondevice may include a plurality of adjacent extraction tanks, eachextraction tank including a screw conveyor operable to deliver solidmaterial from the raw feedstock an a downstream direction and a weiroperable to deliver liquid material from the raw feedstock in anupstream direction.

[0031] The lime input device may be operable to deliver lime to thebiomass either during or after the delivering of the biomass over thegravel layer. The lime pretreatment pile may be maintained at atemperature between approximately 25° C. and 95° C. at ambient pressureand for a time period greater than approximately four weeks. The systemmay include a heat exchanger coupled to the distribution pipe andoperable to control a temperature of the water that is delivered to thedistribution pipe. It may also include an air blower and an airdistribution pipe operable to deliver air to the biomass pile. Acontainer of lime water slurry may be coupled to the air distributionpipe and operable to scrub the air of carbon dioxide before the air isdelivered to the biomass pile. A calcium carbonate input device may beadded to deliver calcium carbonate to the biomass for pretreating thebiomass.

[0032] The system may also include an inoculum input device operable todeliver an inoculum to the biomass pile for fermentation of the biomasspile.

[0033] Yet another embodiment of the present invention relates to asystem for processing biomass, including:

[0034] an end wall;

[0035] a water-impermeable bottom liner;

[0036] a top liner coupled to the bottom liner, the top linerselectively inflatable by one or more fans coupled to the end wall;

[0037] a plurality of water pouches coupled to the top liner, the waterpouches selectively inflatable when the top liner is inflated;

[0038] a gravel layer supported by bottom liner and separated into aplurality of gravel segments;

[0039] a plurality of drain pipes disposed within respective ones of thegravel segments;

[0040] a conveyor belt associated with the end wall and operable todeliver biomass over the gravel segments to form a biomass pile;

[0041] a lime input device operable to deliver lime to the biomass forpretreating the biomass;

[0042] a plurality of distribution pipes coupled to the top liner andassociated with respective ones of the plurality of gravel segments; and

[0043] a plurality of pumps coupled to respective ones of the pluralityof drain pipes and respective ones of the plurality of distributionpipes, the pumps operable to circulate water through the biomass pile bydelivering water to the distribution pipes and receiving water from thedrain pipes after the water has traveled through the biomass pile.

[0044] In more specific embodiments, the biomass may be lignocellulosicbiomass such as bagasse and corn stover. An opening may formed in theend wall for unloading residue left over from the biomass pile afterfermentation. The system may include a sugar extraction device operableto extract sugar from a raw feedstock to produce the biomass. The rawfeedstock may be energy cane or sweet sorghum. The sugar extractiondevice may include a plurality of adjacent extraction tanks, eachextraction tank including a screw conveyor operable to deliver solidmaterial from the raw feedstock an a downstream direction and a weiroperable to deliver liquid material from the raw feedstock in anupstream direction.

[0045] The lime input device may be operable to deliver lime to thebiomass either during or after the delivering of the biomass over thegravel layer. The lime pretreatment pile may be maintained at atemperature between approximately 25° C. and 95° C. at ambient pressureand for a time period greater than approximately four weeks. The systemmay include a heat exchanger coupled to the distribution pipe andoperable to control a temperature of the water that is delivered to thedistribution pipe. It may also include an air blower and an airdistribution pipe operable to deliver air to the biomass pile. Acontainer of lime water slurry may be coupled to the air distributionpipe and operable to scrub the air of carbon dioxide before the air isdelivered to the biomass pile. A calcium carbonate input device may beadded to deliver calcium carbonate to the biomass for pretreating thebiomass.

[0046] The system may also include an inoculum input device operable todeliver an inoculum to the biomass pile for fermentation of the biomasspile.

[0047] Another embodiment of the invention relates to a system forprocessing biomass, including:

[0048] a plurality of geodesic domes arranged in a generally circularpattern, each geodesic dome comprising:

[0049] a water-impermeable bottom liner;

[0050] a top liner coupled to the bottom liner;

[0051] a gravel layer supported by the bottom liner;

[0052] a drain pipe disposed within the gravel layer; and

[0053] a distribution pipe elevated above the gravel layer;

[0054] a plurality of pumps coupled to respective ones of the pluralityof geodesic domes, each pump operable to circulate water through itsrespective geodesic dome by delivering water to the distribution pipeassociated with the respective geodesic dome and receiving water fromthe drain pipe associated with the respective geodesic dome;

[0055] a rotatable conveyor belt surrounded by the geodesic domes andoperable to deliver biomass to each geodesic dome; and

[0056] a lime input device operable to deliver lime to the biomass forpretreating the biomass.

[0057] In specific emobodiments, the biomass may be lignocellulosicbiomass such as bagasse and corn stover. Each top liner may be made of aplurality of hexagonal or pentagonal panels coupled to one another withlips associated with each panel. A foam layer may be coupled to anoutside of the top liner. The lime input device may be operable todeliver lime to the biomass either during or after the delivering of thebiomass over the gravel layer. A calcium carbonate input device may beadded to deliver calcium carbonate to the biomass for pretreating thebiomass.

[0058] Another embodiment of the present invention relates to a systemfor processing biomass, including a fermenter structure configured to:

[0059] accept and store untreated lignocellulosic biomass;

[0060] pretreat the lignocellulosic biomass with lime at a temperaturebetween approximately 25° C. and 95° C. at ambient pressure for a timeperiod greater than four weeks; and

[0061] treat the lignocellulosic biomass with an inoculant.

[0062] One method of the present invention relates to a method ofbiomass pretreatment by adding an alkali to biomass with lignin contentto produce a mixture and incubating the mixture at a temperature betweenapproximately 25° C. and 95° C. at ambient pressure.

[0063] In more specific embodiments, the method also includes incubatingthe mixture for a time period of at least approximately 4 weeks, morespecifically between approximately 4 and 16 weeks. The duration ofincubation may be selected based on incubation temperature. The biomassmay be lignocellulosic biomass such as agricultural waste, bagasse, cornstover and combinations thereof.

[0064] The method may also include circulating water through the biomassduring incubation and circulating air through the biomass duringincubation. The air may be oxygen enriched air. The alkali added mayinclude lime or calcium oxide. When lime is used approximately 0.5 gramsof lime may be added per gram of biomass to produce the mixture, orapproximately 0.1 to 0.5 grams of lime may be added per gram of biomassto produce the mixture. Alternatively, lime may be added to the biomassin an amount between approximately 10% and 30% of biomass by weight.Calcium carbonate may also be added to the mixture.

[0065] The mixture may be incubated at a temperature betweenapproximately 25° C. and 90° C. more specifically between approximately25° C. and 57° C. The incubation temperature may be based on the partialpressure of water at the selected temperature.

[0066] The method may include increasing the enzyme digestibility of thebiomass or producing pulp such as pulp suitable for paper or cardboardproduction.

[0067] The method may also include reducing the lignin content of thebiomass. Lignin content may be reduced by at least 98%, at least 90%, atleast 29%, at least 40%, or at least 67%. Lignin content may be reducedby alkaline oxidation.

[0068] The method may also include fermenting the biomass afterincubation. The may be accomplished by adding an inoculum to themixture. After or during fermentation carboxylate salts may be collectedfrom the mixture.

[0069] The method may additionally include placing the mixture prior toincubation in a storage facility suitable for incubation andfermentation.

[0070] Another method of the present invention relates to a method forproducing enzymatically digestible biomass by adding lime to biomasswith lignin content to produce a mixture, incubating the mixture at atemperature between approximately 25° C. and 55° C. at ambient pressurefor a time period of at least 4 to 16 weeks and circulating waterthrough the mixture during incubation.

[0071] In specific embodiments, air may also be circulated through themixture during incubation. The method may reduce lignin content of thebiomass by at least 67%, or at least 32%. Biomass may be fermented afterincubation.

[0072] Finally, another method of the invention relates to a method forproducing pulp by adding lime to biomass with lignin content to producea mixture, incubating the mixture at a temperature between approximately45° C. and 55° C. at ambient pressure for a time period of approximately10 weeks, and circulating water through the mixture during incubation.

[0073] In more specific embodiments, the method may include circulatingair through the mixture during incubation. The method may reduce lignincontent by at least 90% or by at least 40%. The biomass may be used toproduce paper or cardboard after fermentation.

[0074] For a better understanding of the invention and its advantages,reference may be made to the following description of exemplaryembodiments and accompanying drawings in which like features areindicated by like numbers.

BRIEF DESCRIPTION OF THE DRAWINGS

[0075]FIG. 1 illustrates the results of a prior art study by Chang andHoltzapple showing the enzymatic digestibility of lignocellulose as afunction of lignin content and acetyl content;

[0076]FIG. 2 is a schematic of a system for processing biomass accordingto an embodiment of the present invention;

[0077]FIG. 3 illustrates a fermenter according to an embodiment of thepresent invention;

[0078]FIG. 4 is a cross-sectional view of the fermenter of FIG. 3illustrating a biomass pile therein according to an embodiment of thepresent invention;

[0079]FIG. 5 illustrates a detail of how a geomembrane may be coupled tothe fermenter of FIG. 3 according to an embodiment of the presentinvention;

[0080]FIG. 6 is a schematic of a fermenter layout according to anembodiment of the present invention;

[0081]FIG. 7 is a schematic of a multi-stage countercurrent extractoraccording to an embodiment of the present invention;

[0082]FIG. 8 is a schematic of a screw press with mixing blade accordingto an embodiment of the present invention;

[0083]FIG. 9 is a schematic of a screw mounted at an angle according toan embodiment of the present invention;

[0084]FIG. 10 is a schematic of tanks for use with a horizontal screwaccording to an embodiment of the present invention;

[0085]FIG. 11 illustrates a fermenter according to another embodiment ofthe present invention;

[0086]FIGS. 12A and 12B are various cross-sectional views of thefermenter of FIG. 11 illustrating a biomass pile therein according to anembodiment of the present invention;

[0087]FIG. 13 is a perspective view of water-filed pouches for use inthe fermenter of FIG. 11 according to an embodiment of the presentinvention;

[0088]FIG. 14 is a schematic of a fermenter layout for anotherembodiment of the present invention;

[0089]FIGS. 15A and 15B are top and cross-sectional views, respectively,of a hexagonal panel in a geodesic dome fermenter according to anotherembodiment of the present invention;

[0090]FIG. 16 is a schematic of a pivoting conveyor belt for use in anembodiment of the present invention;

[0091]FIG. 17 is a schematic of a large biomass processing plant withfermenters located in the outposts according to an embodiment of thepresent invention;

[0092]FIG. 18 illustrates an experimental set-up according to anembodiment of the present invention;

[0093]FIG. 19 presents the total mass, holocellulose, lignin and ask fortreatment without air purging at 25° C. according to an embodiment ofthe present invention;

[0094]FIG. 20 presents the total mass, holocellulose, lignin and ask fortreatment without air purging at 50° C. according to an embodiment ofthe present invention;

[0095]FIG. 21 presents the total mass, holocellulose, lignin and ask fortreatment without air purging at 57° C. according to an embodiment ofthe present invention;

[0096]FIG. 22 presents the total mass, holocellulose, lignin and ask fortreatment with air purging at 25° C. according to an embodiment of thepresent invention;

[0097]FIG. 23 presents the total mass, holocellulose, lignin and ask fortreatment with air purging at 50° C. according to an embodiment of thepresent invention;

[0098]FIG. 24 presents the total mass, holocellulose, lignin and ask fortreatment with air purging at 57° C. according to an embodiment of thepresent invention;

[0099]FIG. 25 presents holocellulose loss as a function of ligninremoval for lime pretreatment of bagasse without air purging at 25° C.according to an embodiment of the present invention;

[0100]FIG. 26 presents holocellulose loss as a function of ligninremoval for lime pretreatment of bagasse without air purging at 50° C.according to an embodiment of the present invention;

[0101]FIG. 27 presents holocellulose loss as a function of ligninremoval for lime pretreatment of bagasse without air purging at 57° C.according to an embodiment of the present invention;

[0102]FIG. 28 presents holocellulose loss as a function of ligninremoval for lime pretreatment of bagasse with air purging at 25° C.according to an embodiment of the present invention;

[0103]FIG. 29 presents holocellulose loss as a function of ligninremoval for lime pretreatment of bagasse with air purging at 50° C.according to an embodiment of the present invention;

[0104]FIG. 30 presents holocellulose loss as a function of ligninremoval for lime pretreatment of bagasse with air purging at 57° C.according to an embodiment of the present invention;

[0105]FIG. 31 presents the lignin content in lime-treated bagasse (25°C.) according to an embodiment of the present invention;

[0106]FIG. 32 presents the lignin content in lime-treated bagasse (50°C.) according to an embodiment of the present invention;

[0107]FIG. 33 presents the lignin content in lime-treated bagasse (57°C.) according to an embodiment of the present invention;

[0108]FIG. 34 presents the lignin content in bagasse lime-treatedwithout air purging according to an embodiment of the present invention;

[0109]FIG. 35 present the lignin content in bagasse lime-treated withair purging according to an embodiment of the present invention;

[0110]FIG. 36 presents the lignin conversion of lime-treated bagasse at25° C. according to an embodiment of the present invention;

[0111]FIG. 37 presents the lignin conversion of lime-treated bagasse at50° C. according to an embodiment of the present invention;

[0112]FIG. 38 presents the lignin conversion of lime-treated bagasse at57° C. according to an embodiment of the present invention;

[0113]FIG. 39 presents the lime consumed in treatment of bagasse at 50°C. according to an embodiment of the present invention;

[0114]FIG. 40 presents the lime consumed in treatment of bagasse at 57°C. according to an embodiment of the present invention;

[0115]FIG. 41 presents the lime consumed in treatment of bagasse at 25°C. according to an embodiment of the present invention;

[0116]FIG. 42 presents the 3-day enzyme digestibility of bagasse treatedat 25° C. according to an embodiment of the present invention;

[0117]FIG. 43 presents the 3-day enzyme digestibility of bagasse treatedat 50 ° C. according to an embodiment of the present invention;

[0118]FIG. 44 presents the 3-day enzyme digestibility of bagasse treatedat 57° C. according to an embodiment of the present invention;

[0119]FIG. 45 presents the 3-day enzyme digestibility of bagasse treatedwithout air according to an embodiment of the present invention;

[0120]FIG. 46 presents the 3-day enzyme digestibility of bagasse treatedunder air purging according to an embodiment of the present invention;

[0121]FIG. 47 illustrates a subset of a jacketed reactor system fornon-oxidative lime pretreatment (N₂ supply) according to an embodimentof the present invention;

[0122]FIG. 48 illustrates a subset of a jacketed reactor system fornon-oxidative lime pretreatment (air supply) according to an embodimentof the present invention;

[0123]FIG. 49 presents the particle size distribution of the first andsecond batches of corn stove processed according to an embodiment of thepresent invention;

[0124]FIG. 50 presents profiles of lime consumption for non-oxidativepretreatment at 25, 35, 45 and 55° C. according to an embodiment of thepresent invention;

[0125]FIG. 51 presents profiles of lime consumption for oxidativepretreatment at 25, 35, 45 and 55° C. according to an embodiment of thepresent invention;

[0126]FIG. 52 presents profiles of Klason lignin content innon-oxidative lime pretreatment at 25, 35, 45 and 55° C. according to anembodiment of the present invention;

[0127]FIG. 53 presents profiles of Klason lignin content in oxidativelime pretreatment at 25, 35, 45 and 55° C. according to an embodiment ofthe present invention;

[0128]FIG. 54 presents profiles of acid-soluble lignin content innon-oxidative lime pretreatment at 25, 35, 45 and 55° C. according to anembodiment of the present invention;

[0129]FIG. 55 presents profiles of acid-soluble lignin content inoxidative lime pretreatment at 25, 35, 45 and 55° C. according to anembodiment of the present invention;

[0130]FIG. 56 presents an Arrhenius plot Ink versus 1000/T for theoxidative delignification of corn stover according to an embodiment ofthe present invention;

[0131]FIG. 57 presents composition changes caused by non-oxidative limepretreatment at 55° C. according to an embodiment of the presentinvention;

[0132]FIG. 58 presents sugar yield profiles of untreated corn stoveraccording to cellulose loading rate at the enzyme reaction times: 1, 5,and 72 hours;

[0133]FIG. 59 presents the 3-day enzyme digestibility profiles oftreated corn stover in non-oxidative conditions for 16 weeks at 25, 35,45 and 55° C. according to an embodiment of the present invention;

[0134]FIG. 60 presents the 3-day enzyme digestibility profiles oftreated corn stover in non-oxidative conditions for 1, 2, 4, 8 and 16weeks at 55° C. according to an embodiment of the present invention;

[0135]FIG. 61 presents the 3-day enzyme digestibility profiles oftreated corn stover in oxidative conditions for 16 weeks at 25, 35, 45and 55° C. according to an embodiment of the present invention;

[0136]FIG. 62 presents the 3-day enzyme digestibility profiles oftreated corn stover in oxidative conditions for 1, 2, 4, 8, 12 and 16weeks at (a)25, (b)35, (c)45 and (d)55° C. according to an embodiment ofthe present invention;

[0137]FIG. 63 presents a comparison of the 3-day enzyme digestibilityprofiles between non-oxidative and oxidative treated corn stover for 16weeks at a)25, (b)35, (c)45 and (d)55° C. according to an embodiment ofthe present invention; and

[0138]FIG. 64 presents profiles of protein content reduction duringnon-oxidative and oxidative pretreatments at 55° C. according to anembodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0139] The present invention includes a method of treating biomass,particularly lignocellulosic biomass, with lime or other alkali toproduce useful recovery products. The invention also includes treatmentapparatuses that may be used with the lime treatment method or othertreatment methods.

[0140] The methodology of the present invention includes a process totreat lignocellulosic biomass with lime or other alkali for extendedtime periods to increase enzymatic digestibility. In addition,lignocellulosic biomass can be treated with lime or alkali andcirculated air or oxygen enriched air for extended time periods of time.The methods of the present invention may also be employed to producepulp, including pulp suitable for making paper or cardboard.

[0141] Overall, the processes of the present invention provide veryinexpensive ways to process lignocellulosic biomass. Lime is the leastexpensive alkali and air is free, although circulated or oxygen enrichedair may have some associated coats. The treatment conditions in mostembodiments are very mild (moderate temperatures, 1 atm pressure) soextremely inexpensive vessels may be employed.

[0142] Embodiments of the present invention include pretreatmentprocesses carried out at any of a variety of temperatures ranging from25° C. (ambient temperature in many regions) to 95° C. Although lime isused in many exemplary embodiments of the present invention, otheralkalis including calcium alkalis such as calcium oxide (quick lime) mayalso be suitable.

[0143] Any sort of biomass may be used in the present invention, butlignocellulosic biomass is used in many exemplary embodiments of theinvention. The number of weeks the process is carried out may vary fromapproximately 4-16, depending upon the desired outcome of the processand the temperature at which it operates. Other time periods may also beused to achieve particular results and to accommodate particularconditions, such as starting material, temperature and lime or otheralkali concentration. The process conditions and time period ofoperation to achieve given results for a given starting material will beapparent to one skilled in the art based upon the content of thisdisclosure and knowledge in the field.

[0144]FIG. 1 is taken from Vincent S. Chang and Mark T. Holtzapple,Fundamental Factors Affecting Biomass Enzymatic Reactivity, AppliedBiochemistry and Biotechnology, Vol. 84-86, pp. 5-36. Native herbaceouslignocellulose typically has about 15-20% lignin and woodylignocellulose has about 25-30% lignin. For both herbaceous and woodybiomass, the acetyl content is typically about 3%. FIG. 1 shows thathigh lignin and high acetyl contents reduce enzymatic digestibility.FIG. 1 indicates that reducing the lignin below the native contentsubstantially increases the enzymatic digestibility; however, when thelignin content reaches 10% or less, the enzymatic reactivitysubstantially reaches a plateau. Further lignin removal enhancesreactivity, but not significantly. FIG. 1 shows that when acetyl groupsare removed from the hemicellulose fraction of lignocellulose—forexample, by alkaline treatments—the enzymatic reactivity improves aswell. FIG. 1 indicates that an ideal lignocellulose treatment processshould be capable of removing acetyl groups and also reduce the lignincontent to at least about 10%.

[0145] Although lignin reduction below 10% benefits enzymatic reactivityslightly, the additional cost imposed by further reduction may not bejustified. In contrast, if the goal is to make pulp for paper orcardboard, then it is desirable to remove as much lignin as possible.Ideally for paper, the lignin content is zero, although this usuallyrequires expensive bleaching as a final step.

[0146] The apparati of the present invention include a combined storageand pretreatment systems. Other embodiments include a system alsosuitable for fermentation. The systems include a lined fermentor intowhich untreated biomass may be placed. The untreated biomass may then bepretreated with, for example, lime. Water may be moved through thebiomass pile by an assembly of pumps and pipes that collect water fromthe bottom of the pile and distribute it to the top of the pile. Afterpretreatment is complete, the pile may be subject to further treatment,such as fermentation. Although the primary pretreatment agent isreferred to as lime in the description of apparati, one skilled in theart will understand that other or additional alkali may be used inspecific embodiments in a manner similar to lime.

[0147]FIG. 2 is a schematic of a system 100 for processing biomassaccording to an embodiment of the present invention. In the illustratedembodiment, system 100 includes a water-impermeable bottom liner 102, agravel layer 104, a drain pipe 106, a biomass input device 108, a limeinput device 110, a calcium carbonate input device 112, a distributionpipe 114, a pump 116, a water supply 118, an inoculum supply 120, an airdistribution pipe 122, an air blower 124, a lime water slurry container126, and a heat exchanger 128. The present invention contemplates more,less, or different components for system 100 than those shown in FIG. 2.

[0148] An important advantage of system 100, and other example systemsdescribed below in conjunction with FIGS. 3-17, is that a singlefacility may be utilized to accept and store untreated biomass, pretreatthe biomass, and ferment the biomass, which reduces biomass handling byallowing three operations to be accomplished in a single storagefacility. Solids transport may be accomplished using well establishedtechniques so there is little risk associated with handling biomass.Also, fermentation may occur up to almost a full year, the productconcentration may be very high, thus reducing dewatering costs. Previousbiomass processing systems had to utilize high temperatures and highpressures, which increased the cost of the storage facilities anddecreased the quality of the product obtained.

[0149] Liner 102, which may be formed from any suitablewater-impermeable material, functions to support gravel layer 104 andprevent any water or other material from entering the ground. Althoughliner 102 may be placed upon any suitable support, it is preferable thatliner 102 lie in a suitable pit or bermed wall in the ground. Liner 102may have any suitable shape and the depth of liner 102 should besuitable to handle a desired amount of gravel for gravel layer 104. Anexample depth for gravel layer 104 is approximately three feet; however,other suitable depths may also be utilized for gravel layer 104. Gravellayer 104 is comprised of any suitable loose or unconsolidated depositof rounded pebbles, cobbles, boulders, or other suitable stone-likematerial that functions to allow water to flow relatively freelytherethrough.

[0150] On top of gravel layer 104 is a biomass pile 105 that isdelivered over gravel layer 104 via biomass input device 108. Biomassinput device 108 represents any suitable device for creating biomasspile 105, such as a suitable conveyer system, front end loader, or othersuitable delivery system. As described above, the biomass formingbiomass pile 105, in one embodiment, is lignocellulosic biomass, such asbagasse, corn stover, or other suitable biomass.

[0151] Lime input device 110 and calcium carbonate input device 112 areany suitable devices operable to deliver lime and calcium carbonate,respectively, to the biomass as biomass pile 105 is being formed. Inother embodiments, the lime and/or calcium carbonate is delivered afterbiomass pile 105 is formed. As described above, lime is utilized topretreat the biomass and, in some embodiments, calcium carbonate 112 mayalso be used to pretreat the biomass. Although the amount of lime addedto biomass pile 105 may vary depending on the type of biomass, in oneembodiment, an amount of lime delivered to biomass pile 105 is betweenapproximately 10% and 30% of the biomass by weight.

[0152] Water from water supply 118 is circulated through biomass pile105 by pump 116 by delivering the water through distribution pipe 114,which may be any suitable perforated conduit and is elevated abovebiomass pile 105, and recovering the water through drainpipe 106 afterit has traveled through biomass pile 105 and gravel layer 104.Circulation may either be continuous with a relatively low flow rate ormay be intermittent with a relatively high flow rate. With a continuouscirculation and low flow rate, channeling may occur which is undesirablebecause some portions of biomass pile 105 may not be wetted. Unevenwetting of biomass pile 105 may cause the following problems: incompletepretreatment of biomass pile 105, poor temperature control, andspontaneous combustion of dried portions of biomass pile 105. Anintermittent circulation and high flow rate periodically floods biomasspile 105, thus ensuring all or most portions are wetted, therebyovercoming the potential problems of continuous circulation with lowflow rate.

[0153] The temperature of the water circulated through biomass pile 105may be regulated by heat exchanger 128. Heat exchanger 128 may be anysuitable device used to control the temperature of the water circulatedthrough biomass pile 105. For example, heat exchanger 128 may be ashell-and-tube type heat exchanger.

[0154] While biomass pile 105 is being pretreated, air may be blownupward through biomass pile 105 to enhance lignin removal by alkalineoxidation. This may be facilitated by air blower 124 forcing air throughair distribution pipe 122, which may be any suitable perforated conduitdisposed proximate gravel layer 104. Because air contains carbondioxide, it may react with lime to form calcium carbonate, anunproductive reaction. To prevent this from occurring in biomass pile105, the air may be scrubbed of carbon dioxide by passing it throughlime water slurry in container 126, which may be a suitable packedcolumn or tank. Oxygen enriched or may also be used.

[0155] As described above, biomass pile 105 may be subject to afermentation process while disposed over gravel layer 104. To facilitatethe fermentation after pretreatment is complete, water is circulatedthrough biomass pile 105 that contains an inoculum of acid-formingmicroorganisms obtained from inoculum supply 120. The acid-formingmicroorganism start to degrade biomass pile 105 forming carboxylic acidsthat react with calcium carbonate to form calcium carboxylate salts.Water may then be circulated through biomass pile 105 to remove thecarboxylate salts.

[0156] The storage, pretreatment, and fermentation of biomass may alsobe accomplished using other suitable storage facilities or systems.Various embodiments of these systems are described below in conjunctionwith FIGS. 3-17.

[0157]FIGS. 3 and 4 are perspective and cross-sectional views,respectively, of a system 200 for storing, pretreating, and fermentingbiomass in accordance with another embodiment of the invention. System200 is similar to system 100 in FIG. 2; however, system 200 includes ageomembrane 202 coupled to a grid-like lattice structure 204 to form aroof for the facility. In the illustrated embodiment, grid-like latticestructure 204 is formed from any suitable structural beams, such asI-beams, and has any suitable shape, such as a half cylinder shape, anarch, or other shapes suitable to form an enclosure between geomembrane202 and a bottom liner 206 that supports a gravel layer 208.

[0158] Grid-like lattice structure 204 includes a conveyer belt 210coupled thereto and running along the length of grid-like structure 204to deliver biomass within the enclosure and over gravel layer 208. Anysuitable conveyer system is contemplated by the present invention forconveyer belt 210. In addition, conveyer belt 210 may be coupled togrid-like lattice structure 204 in any suitable manner.

[0159] Geomembrane 202, which may be formed from any suitable material,may be coupled to grid-like lattice structure 204 in any suitablemanner; however, one embodiment of coupling geomembrane 202 to grid-likelattice structure 204 is illustrated below in conjunction with FIG. 5.Referring to FIG. 5, one or more bolts 212 are utilized to couplegeomembrane 202 to grid-like lattice structure 204. To prevent thecorrosion of bolts 212 or grid-like lattice structure 204, a boot 214formed from the same or similar material as geomembrane 202 is utilizedto cover bolts 212. Other suitable fasteners other than bolts may alsobe utilized to couple geomembrane 202 to lattice structure 204. A pairof stiffener plates 218 may provide stiffness to geomembrane 202, whichis disposed between stiffener plates 218 and lattice structure 204 andcoupled therebetween by bolts 212.

[0160] Also illustrated in FIG. 5 is a foam layer 216 coupled to anoutside surface of geomembrane 202. Any suitable foam material may beutilized for foam layer 216 and it may be coupled to an outside surfaceof geomembrane 202 using any suitable method, such as a spray-in-placemethod. Foam layer 216 functions to make the exterior somewhat rigid toprevent geomembrane 202 from flexing in the wind, which may lead topossible fatigue failure. Although not illustrated, foam layer 216 maybe painted or otherwise coated with a suitable coating to resist UVdamage.

[0161]FIG. 6 illustrates a plan view of system 200 according to anembodiment of the invention. A plurality of pumps 220 are suitablylocated adjacent system 200 to pump clear water from clear water supply222 through a suitable conduit system to distribution pipes 224 coupledto geomembrane 202 and that are operable to direct the water towardsbiomass pile 205. A plurality of drain pipes 226 associated withrespective distribution pipes 224 may be utilized to collect the waterafter it has traveled through biomass pile 205 and be recirculated bypumps 220. A small side stream, as denoted by reference number 228, maybe pumped from each pump 220 to its adjacent pump 220.

[0162] In one embodiment, clear water from clear water supply 222 isintroduced to one end of system 200, thereby establishing aconcentration gradient along biomass pile 205. A portion of biomass pile205 with the most dilute carboxylate salts reacts more rapidly becausethere is less inhibition. Eventually, the entire biomass pile 205 isreacted. By adjusting the rate water is pumped to adjacent pumps 220,the reaction rate may be regulated so that the reaction is completed afew weeks prior to harvesting the next season's crop. Solid residuesthat remain in the enclosure (for example, lignin, unreactedcarbohydrates, may be removed using front-end loaders, dump trucks, orother suitable devices). After fermentation of biomass pile 205, theresulting products, as represented by concentrated fermentation broth230 in FIG. 6, may be removed using pumps 220.

[0163] Also illustrated in FIG. 6 is a system 232 for delivering biomassto conveyer belt 210 according to one embodiment of the invention. Inthe illustrated embodiment, system 232 includes a grinder 234 and asugar extraction device 236. Grinder 234 receives a suitable feedstock,such as raw energy cane, and processes it before delivering it to sugarextraction device 236. Feedstock other than raw energy may be alsoutilized, such as high-yield sweet sorghum. To make best use of thesugars in the feedstock, the sugars may be extracted and sold for foodor as feedstock for pure-culture fermentations (for example, ethanol,and citric acid). Grinder 234 may be any suitable grinder, such as ahammer mill, operable to grind raw feedstock. Sugars are then extractedusing sugar extraction device 236.

[0164] Sugar extraction device 236 may be a conventional sugar mill thatuses high pressure rollers to squeeze sugars out of energy cane. Sugarcane varieties with high sugar concentration may be employed to maximizethe amount of sugar produced from each roller. Wash water may becirculated through sugar extraction device 236 in order to extract sugarwater therefrom. The feedstock coming out of sugar extraction device 236is the biomass that is delivered to system 200 using conveyer belt 210or other suitable delivery system.

[0165] To reduce the cost of extracting sugars from raw feedstock, alow-cost method is desirable. An example low-cost method is illustratedbelow in conjunction with FIG. 7, which shows a multi-stagecountercurrent sugar extractor 300 according to one embodiment of theinvention. The larger arrows 302 illustrate solids flow and the smallerarrows 304 illustrate liquid flow. Extractor 300 includes a plurality ofadjacent extraction tanks 306, wherein each extraction tank 306 includesa screw conveyer 308, as illustrated in FIG. 8, and a weir 310, asillustrated in FIG. 9.

[0166] Referring to FIGS. 7 and 8, a feedstock slurry with a high watercontent is disposed within extraction tank 306. Screw conveyer 308,which may be any suitable conical screw conveyer, transports the slurryupward in the expanding cone of conveyer 308. This allows less room,which forces water out of the slurry causing it to exit through theperforated pipe 312 of conveyer 308 and back down towards the interiorof extraction tank 306.

[0167] To achieve mixing in the high water slurry, a mixer blade 314 maybe employed on the end of shaft 316 of conveyer 308. This allows asingle motor 318 to drive both mixer blade 314 and the conical portionof screw conveyer 308, which saves capital costs. Weir 310 is protectedfrom the agitation resulting from mixer blade 314, thereby allowing thebiomass to settle so liquid selectively flows over weir 310 to thepreceding extraction tank 306. In one embodiment, a screen (not shown)is employed on weir 310 to filter out solids. To prevent possibledegradation of sugars in extraction tank 306, lime may be added tomaintain a sufficiently high pH so that microorganisms cannot grow. Totake advantage of the mixing, all the fermentation lime and calciumcarbonate may be added in the last extraction tank 306 prior todischarging the solid biomass.

[0168] Referring to FIG. 9, the water flow is represented by arrow 304and the solids flow is represented by 302. The adjacent extraction tanks306 are not illustrated in FIG. 9 for clarity of description purposes.Conveyer 308 is tilted at a suitable angle in order to facilitate thedelivering of the solids to the downstream extraction tanks 306.

[0169]FIG. 10 is a schematic of extraction tanks 306 according toanother embodiment of the present invention. FIG. 10 illustrates how thescrew conveyers 308 may be mounted horizontally in extraction tanks 306to achieve the countercurrent flow of solids and liquids. As illustratedin FIG. 10, the large arrows 330 illustrate solids flow while the smallarrows 332 illustrate the liquid flow over weirs 310. Multiple screwsconveyors 308 may be located in a single extraction tank 306, thusgiving a large perforated surface area through which the water mayeasily flow. One advantage of the horizontal configuration for conveyers308 in FIG. 10 is that a single motor (not explicitly shown) may servicemultiple extraction tanks 306, thus reducing capital costs.

[0170]FIG. 11 is a perspective view and FIGS. 12A and 12B are variouscross-sectional views of a system 400 for storing, pre-treating, andfermenting biomass in accordance with another embodiment of theinvention. System 400 is similar to system 200 described above; however,system 400 includes an end wall 402, which may be any suitable rigidstructure, having one or more fans 404 that are operable to selectivelyinflate a top liner 406 having a plurality of selectively inflatablepouches 408 coupled thereto. In this manner, top liner 406 may be in adeflated state when not in use and, when desired to store, pre-treat andferment biomass therein, top liner 406 may be inflated by fans 404 toform an enclosure for the biomass. Top liner 406 may be formed from anysuitable inflatable material, such as plastic, which functions toexclude rain water and to maintain anaerobic conditions within theenclosure. To prevent top liner 406 from deflecting in the wind, pouches408 are filled with water or other suitable liquid using any suitableconduit system. Details of pouches 408 are illustrated below inconjunction with FIG. 13.

[0171] Referring to FIG. 13, a portion of top liner 406 is illustratedwith some of pouches 408. Suitable conduits 410 are illustrated asdelivering water or other liquid to and from pouches 408 in order toinflate or deflate pouches 408 as desired. Pouches 408 may be formedfrom any suitable inflatable material and may be formed with anysuitable configuration and arrangement.

[0172] Referring back to FIGS. 11, 12A and 12B, end wall 402 alsoincludes a conveyor port 412 that functions to accept a suitableconveyor system for delivering the biomass to the inside of thestructure. Inside the structure is a suitable gravel layer 414 that is,in the illustrated embodiment, divided into a plurality of segments.Each of these segments includes a drain pipe 416 and a distribution pipe418 that are coupled to a suitable pump 420 for the purpose ofcirculating water through biomass pile 405. Also illustrated in FIG. 11is a truck door 422 suitable for allowing end loaders or other suitableequipment to remove the residue left over after the fermentation ofbiomass pile 405.

[0173]FIG. 14 is a schematic of a system 500 for processing biomassaccording to one embodiment of the invention. In the illustratedembodiment, system 500 includes a plurality of geodesic domes 502arranged in a generally circular pattern, wherein each geodesic dome 502includes similar system components to those illustrated above inconjunction with systems 100, 200, and 400. As illustrated in FIGS. 15Aand 15B, the roof of each geodesic dome 502 is constructed from aplurality of panels 504 having stiffening ribs 506 and a lip 508 forcoupling panels 504 to one another. Panels 504 may be any suitableshape, such as hexagonal or pentagonal, and may be formed from anysuitable material. Panels 504 may also be coupled to one another alonglips 508 using any suitable method, such as plastic welding.

[0174] Referring back to FIG. 14, a biomass delivery system 510, withsimilar components to those described above in conjunction with FIG. 6,delivers biomass to a pivoting conveyor belt 512, as shown below inconjunction with FIG. 16. Any suitable rotatable conveyor belt systemmay be utilized for conveyor belt 512. Conveyor belt 512 is surroundedby geodesic domes 502 and functions to deliver biomass to each of thegeodesic domes 502. Each geodesic dome 502 may have a hole near the topinto which the biomass enters. Once the biomass pile is built, then thehole may be closed using any suitable method. Foam (not shown) may alsobe coupled to an exterior of geodesic domes 502 to provide stiffness,plug holes, and protect the tops of the geodesic domes from theenvironment. One advantage of the embodiment illustrated in FIG. 14 isthat multiple individual facilities give greater flexibility whenscheduling filling, pre-treatment, fermentation, and emptying ofbiomass.

[0175]FIG. 17 illustrates a system 600 processing biomass according toanother embodiment of the present invention. FIG. 17 illustrates thatthe shipping distance of raw biomass to a central plant 602 may bereduced by connecting a plurality of outposts 604 to central plant 602via suitable conduits, such as pipelines. Each outpost 604 would includethe components illustrated in any of the systems described above. Duringthe harvest season, the pipelines would shift sugar water to centralplant 602 for purification. Once the biomass pile has been built and thepre-treatment is complete, then the pipeline may be used to shipfermenter broth solutions to central plant 602 for concentration andconversion to useful products, such as ketones, alcohols, and carboxylicacids.

[0176] The following examples are included to demonstrate specificembodiments of the invention. Those of skill in the art should, in lightof the present disclosure, appreciate that many changes may be made inthe specific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

EXAMPLES Example 1 General Pretreatment Conditions

[0177] Specific embodiments of the present invention includelignocellulosic biomass treatment with lime only or lime with circulatedair, including oxygen enriched air. Such embodiments may be used totreat, for instance corn stover and bagasse. The general conditions ofthese embodiments are as follows:

[0178] Pressure: 1 atm or ambient pressure to avoid the need forpressure vessels.

[0179] Temperature: Temperatures ranging from 25 to 57° C. As expected,lignin removal is more rapid at higher temperatures. In principle, thereaction could be operated as high as 100° C. and the pressure wouldremain 1 atm. However, at 100° C, the partial pressure of water would be1 atm and the partial pressure of air would be 0 atm. In this case, thebenefits of oxidizing the lignin could not be realized. Therefore, it isadvisable to reduce the temperature to reduce the water partialpressure. The following table provides guidance in temperatureselection: Temperature (° C.) Water Partial Pressure (atm) 50 0.121 600.197 70 0.308 80 0.468 90 0.692 95 0.834

[0180] In an exemplary embodiment, 90° C. is the upper temperature limitbecause above this temperature the partial pressure of air is too lowfor effective lignin degradation.

[0181] Lime Loading: Lime is consumed due to reactions within thebiomass and it also reacts with carbon dioxide in the purged air.However, in most embodiments, a lime loading of 0.5 g Ca(OH)₂/g biomassis sufficient to obtained desired pretreatment outcomes. Lime loadingcan be lowered to about 0.1 to 0.35 g Ca(OH)₂/g biomass, depending onthe time and temperature. Lime only treatment (without circulated air)is not optimal for making pulp because lignin removal is not sufficient,however it may be more than sufficient for preparing biomass for laterenzymatic digestion. The advantage of lime only pretreatment is thatlime consumption is generally less than in the lime with circulated airpretreatment embodiments. Also, the expense of air addition iseliminated.

[0182] Time: To enhance biomass digestibility, the following times aregeneral guidelines: Temperature (° C.) Time (weeks) 25 16 35 16 45 8 554

[0183] These time/temperatures are guidelines, not firm requirements andmay be varied depending upon other reaction conditions, such as pressureand lime loading.

[0184] To produce pulp for paper or cardboard, more lignin is preferablyremoved than when enhancement of enzymatic digestibility is the desiredoutcome of the process. For production of pulp, the following times aregeneral guidelines: Temperature (° C.) Time (weeks) 45 >10 55 >10

[0185] These time/temperatures are guidelines, not firm requirements andmay be varied depending upon other reaction conditions, such as pressureand lime loading.

[0186] Air: Access to circulated air and hence oxygen in a biomass pileis limited. However, the presence of circulated air or oxygen enrichedair (including pure oxygen) significantly enhances the removal oflignin. Therefore, in some embodiments of the present invention, thebiomass pile is supplied with circulated air or oxygen enriched air(often simply referred to as “air”). Previous studies show that pureoxygen treatment is only slightly better than ambient air attemperatures near 50° C. At higher temperatures (e.g., >80° C.) pureoxygen may have significant advantages over ambient air alone becausethe nitrogen in the air reduces the partial pressure of oxygen. Limeonly treatment without ambient air or oxygen enriched air alsosignificantly increases the enzymatic digestibility of biomass, althoughnot as much as when air is supplied.

Example 2 Preliminary Experiments to Determine Process Conditions andtheir Effects

[0187] Biomass delignification by lime treatment occurs very quickly athigh-temperature and high-pressure oxygen conditions (Chang, S. “LimePretreatment of Lignocellulosic Biomass”, Ph. D. Dissertation, Texas A&MUniversity, May 1999). To determine whether long-term delignificationtreatment was feasible, an experiment was conducted in which sugarcanebagasse underwent lime pretreatment using air purging at temperatureslower than 60° C.

[0188] The dry weight of raw sugarcane bagasse (35 mesh) was determinedusing the NREL Standard Procedure No. 1 (NREL (1992). Chemical Analysis& Testing Standard Procedure, National Renewable Energy Laboratory,Golden, Colo.). Several 125-mL Erlenmeyer flasks were loaded with 3 gdry weight of sugarcane bagasse, 1.5 g of Ca(OH)₂ (50% loading) (FisherScientific Co.) and 27 mL of distilled water. Several flasks used air asthe oxygen source. An equal number of flasks had no air contact and weresimply capped as a control. As shown in FIG. 18, the flasks exposed toair were equipped with appropriate 2-hole rubber stoppers through whichtwo glass tubes served as inlet and outlet for the process.

[0189] An incubator equipped with a shaker was used to incubate thesamples at the following temperatures: 57° C., 50° C. and 25° C. (roomtemperature). At appropriate times, flasks were removed and analyzed forlignin content, 3-day cellulase enzyme digestibility, total mass loss,and lime consumption. Thus, these parameters were measured as a functionof time.

[0190] The detailed procedure for the process follows:

[0191] 400 g of 40-mesh untreated bagasse was placed in several 2 Lcentrifuge bottles. About 500 mL of water was added to each centrifugebottle, which were then stirred for about 15 minutes. The bottles werecentrifuged at 3500 rpm or more for 5 minutes. As much water as possiblewas decanted, then the bagasse was re-washed according to the aboveprocedure until the water decanted did not appear to be any clearer thanin the previous cycle.

[0192] The contents of the centrifuge bottles were transferred intoother containers and dried at 45° C. for 24 hours or longer ifnecessary. The dry biomass was allowed to regain equilibrium moisturecontent with the environment, which in some cases took several days.After equilibrium was obtained, the moisture content of the samplebiomass (X₁) was obtained as described in NREL Standard Procedure No.001.

[0193] Several experimental flasks were prepared. Each was filled with 3g dry weight of the biomass, 1.5 g of Ca(OH)₂ and 27 mL of distilledwater. The exact amount of biomass (W₁) and lime (W_(initial)) added toeach flask was recorded to the nearest 0.001 g.

[0194] Flasks were placed in a shaking incubator at the appropriateexperimental temperature. Duplicate flasks were prepared for each set ofexperimental conditions. These flasks were later divided into identicalsample sets A and B. Flasks were removed from the incubator only whennecessary to monitor the pretreatment process as described below.

[0195] Flasks belonging to sample set A were tested for lime consumptionas a function of time. For each flask, after removal from the incubator,the contents were transferred to beaker. As much water as necessary wasused to recover as much of the biomass from the flask as possible.

[0196] Hydrochloric acid was added to the beaker using a titrationapparatus. The buret in the apparatus was filled with a certifiedstandard solution of 1 N hydrochloric acid to a starting volume(V₁). Thebiomass solution in the beaker was titrated to a pH of between 6.80 and7.00. The final volume of HCl (V₂) was recorded and used to calculatethe amount of line remaining in the biomass sample as follows:$W_{remaining} = {\frac{1\quad {mol}\quad {of}\quad {{Ca}({OH})}_{2}}{2\quad {mol}\quad {HCl}} \times \frac{N_{HCl}\left( {V_{1} - V_{2}} \right)}{100} \times {MW}}$

[0197] where,

[0198] W_(remaining)=Total amount of lime remaining in the biomasssample(g),

[0199] N_(HCl)=Normality of the certified standard HCl solution (mol/L),

[0200] V₁=Starting volume of HCl in titration (mL),

[0201] V₂=Final volume of HCl in titration (mL).

[0202] MW=Molecular weight of lime (74.092 g/mol)

[0203] Using the exact amount of lime added to the samples beforepretreatment (W_(initial)) and the amount remaining afterwardsW_(remaining), the amount of lime consumed during pretreatment wascalculated as follows:

Amount of lime consumed (g/g dry biomass)=W _(initial) −W _(remaining)

W ₁×(1−X ₁)

[0204] The remainder of the biomass was washed as describe above thenstored for use in a 3-day enzyme digestibility analysis.

[0205] Flasks belonging to sample set B were tested for biomass weightloss due to pretreatment.

[0206] After removal of the sample flasks from the incubator, aceticacid was added to each to reduce the pH to approximately 5-6 andsolubilize any unreacted lime. The contents of each flask was thentransferred to a 2 L centrifuge bottle, using as much water as necessaryto ensure transfer of as much treated biomass as possible. Thecentrifuge bottle was then filled with water and stirred for 15 minutes.Next the water/biomass mixture was centrifuged at 3500 rpm or more for 5to 10 minutes.

[0207] A vacuum filtration apparatus using a Buchner funnel and apredried preweighed filter paper was prepared. As much water waspossible was decanted from the centrifuged samples into the vacuumfiltration apparatus. The washing and filtering process was repeateduntil the filtrate became clear. Filter papers were replaced asnecessary.

[0208] After washing, as much biomass as possible, using as much waterwas necessary, was transferred to a beaker. The biomass and all filterpapers used during its washing were dried at 45° C. for 24 hours orlonger. The biomass and filters were then cooled in a desiccator untilthey reached room temperature. Then the net weight of the biomass wasobtained ( W₂).

[0209] Immediately after weighing, about 0.5 g of the dried biomass wasused to determine the moisture content (X₂) as described in the NRELStandard Procedure No. 001. The remainder of the biomass was stored foruse in a 3-day enzyme digestibility analysis.

[0210] The total weight loss due to pretreatment was calculated usingthe following formula:${{Total}\quad {Weight}\quad {Loss}\quad \%} = \frac{{W_{1} \times \left( {1 - X_{1}} \right)} - {W_{2} \times \left( {1 - X_{2}} \right)}}{W_{1} \times \left( {{1 - X},_{1}} \right)}$

[0211] where,

[0212] W₁=Weight of the washed raw biomass before pretreatment in eachflask (g),

[0213] X₁=Moisture content of the washed raw biomass at room temperature(g H₂O/g total weight),

[0214] W₂=Weight of the dried biomass, and

[0215] X₂=Moisture content of the dried biomass (W₂).

[0216] Remaining biomass from matching flasks of sample sets A and Bwere combined for a 3-day enzyme digestibility analysis.

[0217] The Klason lignin content of the pooled samples was determinedusing NREL Standard Procedure No.003. Using the same procedure, the ashcontent in the biomass was also determined. Assuming that baggase iscomposed only of lignin, ash, and holocellulose, the holocellulosecontent was also obtained by subtracting ash and lignin contents from100%.

[0218] The procedure used for the 3-day digestibility studies wasidentical to the procedure in Sushien Chang's dissertation (Texas A&MUniversity, 1999) under the title “Enzymatic Hydrolysis Procedure forFundamental Studies of Lime Pretreatment.”

[0219] In the standard analysis procedure, 2.5 g dry weight biomass isused as a sample. If other weights were used, normally because 2.5 g ofbiomass was not available after pretreatment, amounts of all reagentswere adjusted in proportion to the actual amount of biomass.

[0220] The final samples were analyzed for glucose and xyloseconcentration using an HPX-87P carbohydrate HPLC column (BioradLaboratories). The final results were reported in grams of sugar yielded(glucose+xylose) per gram dry weight of untreated biomass. This data maybe obtained from the raw glucose and xylose concentration data bymultiplying the result, which is in grams of sugar yielded per gram dryweight of treated biomass by the dry weight of biomass remaining afterwashing (W₁×(1−X₂)) and then dividing by the total dry weight ofuntreated biomass (W₁×(1×X₁)).

[0221] This procedure assumes that any water-soluble substancesresulting from the pretreatment are not digestible by cellulase enzyme.

[0222] FIGS. 19 to 28 depict the total mass, holocellulose, lignin andash in each sample treated and analyzed as described above as functionof time.

[0223]FIGS. 23-28 show that, for all experimental conditions in whichlime is supplied, there is a rapid decrease of holocellulose and ligninin the first 7 days. After the first 7 days, the material loss begins tolevel off. A more rapid material loss was observed if the temperaturewas higher (FIGS. 25 and 28).

[0224] In the samples without air purging, after the initial materialloss, no significant loss occurred (FIGS. 23, 24 and 25). In samplessubjected to air purging (FIGS. 26, 27 and 28), material loss continues,although the rate of degradation is lower than during the first month.Also, selective lignin removal can be observed in these samples, with amore rapid removal at higher temperatures.

[0225] Selective lignin removal is significant because it describes theeffectiveness of some embodiments of the present invention. Ideally, agood delignification process should remove lignin without a significantloss of holocellulose.

[0226] FIGS. 29 to 34 show holocellulose loss as a function of ligninremoved.

[0227] The slopes from the linear regressions in FIGS. 29 to 34 indicatethe selectivity of the process. The selectivity, defined as g ofholocellulose lost/g of lignin removed, is ideally as low as possible.Table 1 presents the selectivities (slopes) of the linear regressionsfrom FIGS. 29 to 34 TABLE 1 Selectivity of holocellulose loss againstlignin removal (g holocellulose/g lignin) Temp. (° C.) ±(95% C.I.) NoAir ±(95% C.I.) Air 25 1.032 0.078 0.746 0.112 50 0.905 0.107 0.6980.096 57 0.857 0.110 0.724 0.151

[0228] The results of the experimental samples not provided with airsuggest that the selectivity decreases with temperature. In the case ofexperimental samples provided with air it appears that there is nodifference in selectivity based on temperature. When comparing thesamples provided with and air and those without air that were incubatedat the same temperature, the 95% confidence intervals suggest that theselectivity is smaller (better), for the samples provided with air forboth 25° C. and 50° C., but there is no significant difference for 57°C.

[0229]FIGS. 35-39 present lignin content of the experimental samples,expressed as g of lignin remaining/100 g of treated bagasse.

[0230]FIGS. 36-39 suggest that delignification is directly related totemperature and the presence of oxygen. FIGS. 36 and 37 show thatdelignification was more pronounced when oxygen was present. FIG. 34shows that when oxygen is not present, temperature does not have asignificant effect on delignification. On the other hand, in FIG. 35,where oxygen was present, delignification decreased with temperature.

[0231] Even when there is no oxygen present (FIGS. 36-38),delignification occurs very rapidly during the first week and continuesto level off after about a month. Because the samples that were notprovided with air were in capped bottles, these bottles contained a headof air, which could provide some oxygen and give a high delignificationrate during the first week. To test this hypothesis, a sample was firstpurged with nitrogen for 10 minutes and then capped. The result is shownin FIG. 33. Delignification rate of the purged samples was similar tothe capped bottle samples, indicating that the small amount of oxygen inthe head space of capped bottles is insignificant. Therefore, it islikely that some of the lignin in the bagasse is labile to lime aloneand does not require oxygen for its degradation.

[0232] Another way of analyzing lignin removal is by examining thefraction of lignin removed or lignin conversion as a function of time,which is computed as follows:${{{Lignin}\quad {Conversion}} = \frac{L_{0} - L_{t}}{L_{0}}},$

[0233] where L₀=lignin content at time 0, and

[0234] L_(t)=lignin content at time t.

[0235]FIGS. 40-42 show that without air, lignin conversion is only 20 to30%, whereas with air purging, lignin conversion increases significantlyat higher temperatures to over 70%.

[0236]FIGS. 43-45 show the estimated lime consumption during biomasspretreatment. Those samples that were subject to air purging wereexposed to carbon dioxide in the air. Because the pretreatment takesseveral months to complete, the amount of carbon dioxide that reactswith the lime was significant, thus the lime consumption obtained inthis experiment is an overestimate. The avoid this higher limeconsumption, the air may be scrubbed to remove carbon dioxide prior toaddition to the biomass.

[0237]FIGS. 43 and 44 show that the consumption of lime as a function oftime is linear at experimental temperatures of 50° C. and 57° C. Theslopes of the curves were 1.606×10⁻³±0.125×10⁻³ (95% confidenceinterval) g of Ca(OH)₂/(g Of untreated biomass·day) for the treatment at50° C. and 1.839×10⁻³±0.132×10⁻⁻³ (95% confidence interval) g ofCa(OH)₂/(g of untreated biomass·day) for the treatment at 57° C.

[0238] The experiments without addition of air did not show anysignificant lime consumption after the first week.

[0239] In FIG. 41, it can be observed that the consumption of lime inthe biomass sample climbed significantly after the flow of air wasincreased, showing that the carbon dioxide in the air did consume thelime.

[0240] Iogen cellulase enzyme (Iogen Laboratories), with an averageactivity of 67.9 FPU/mL, was used to run 3-day cellulase enzymedigestibility. (See FIGS. 46-50).

[0241] FIGS. 46 to 48 show that to enhance enzymatic digestibility,pretreatment after 14 to 21 days is unnecessary. FIGS. 49 and 50 showthat higher temperatures achieve higher conversions, even for thesamples without added air (FIG. 46).

Example 3 General Conditions and Methods

[0242] The following conditions and methods were used in the experimentsof Example 4 and may be readily adapted by one of skill in the art todetermine other suitable embodiments of the present invention.

[0243] Particle Size Distribution of Raw Biomass

[0244] Sieves

[0245] USA standard testing sieves (A.S.T.M.E.-11 Specification) TABLE ASpecification of Sieves Tyler Equivalent Opening size Sieve number Meshmm in 4 4 4.750 0.1870 20 20 0.850 0.0331 30 28 0.600 0.0234 40 35 0.4250.0165 50 48 0.300 0.0117 80 80 0.180 0.0070 100 100 0.150 0.0059

[0246] Procedures

[0247] 50 g dry biomass was loaded on the sieve of mesh No. 100. Thelid, bowl and seive apparatus was vigorously shaken in a horizontalplane for 1 minute. The particles collected in the seive were stored.Particles in the bowl where transferred to a seive of lower mesh numberand the process was repeated until mesh No. 4 was reached. All samplesfrom seives were dried at 105° C. for 24 hours, then weighed todetermine dry weight for particles collected by each seive size.

[0248] Lime Pretreatment

[0249] Lignocellulosic substrate was pretreated with lime in the presentof water. Four sets of packed bed PVC columns (D×L=1 inch×17 inches)were used for the lime-pretreatment reaction at 25 (ambienttemperature), 35° C., 45° C., and 55° C. Each set included two subsets,one with and one without aeration to achieve oxidation and non-oxidationconditions, respectively. The total number of columns for each subset is10 in order to allow analysis at five different run-times. Three sets ofcolumns with water jackets were operated at three differenttemperatures, 35° C., 45° C., and 55° C., by the water heating andcirculating system.

[0250] The water heating and circulating system had two parts: atemperature controller and a water circulator. (See FIGS. 51 and 52.)The temperature controller contained a temperature controller ({fraction(1/16)} DIN, OMEGA), a thermocouple (KTSS-18G-18, OMEGA), a heatingelement (1.5 kW, 120 V), a solid-state relay (RSSDN-25A, Idec Co.),fuses (12.5 A and ¼ A), and a main switch. The water circulatorcontained a centrifugal pump (¾ HP, TEEL), a water tank (8 gal, NalgeneCo., USA), a manifold having one input and 20 output fittings, andreturn pipelines.

[0251] Air supplied by the Carter-Mattil compressor was preheated andsaturated in the cylinder within the water tank and then distributed toeach column by the air-manifold having one input and ten outputfittings. Compressed nitrogen gas (Plaxair Co., College Station, Tex.)was used to make the non-oxidation condition and supplied to each columnby the N2-manifold after preheating and saturation. (See FIG. 47.)

[0252] Fill water into the water tank over the level of the heatingelement. Turn on the centrifugal pump to circulate water. Fillsufficient water into the tank up to top level.

[0253] For pretreatment, water was placed in the water tank to cover theheating element. The centrifugal pump was activated to circulate thewater and then the tank was filled to top level. The temperaturecontroller was used to heat water to the selected pretreatmenttemperature and the entire heating and circulating system was allowed toreach a steady state. (This was not required for pretreatments atambient temperature.)

[0254] Raw biomass (15.0 g dry weight of corn stover), lime (7.5 g dryweight), and distilled water (150 mL) were transferred into the reactorafter thoroughly being mixed using a spatula.

[0255] The biomass mixture was transferred to the reactor, which wastightly capped. A bubble indicator filled with 20-25 mL of distilledwater in a 50 ml plastic tube was used to measure the gas flow rate.

[0256] A main valve was slowly opened to supply either nitrogen fornon-oxidation pretreatment or air for oxidation pretreatment separately.Bubble formation was confirmed in the bubble indicator. The gas flowrate was adjusted to 1 bubble/second using a clamp, which was placed atthe air intake tube in the bottom of the reactor.

[0257] Gas pressure (4.5-5.0 psi in case of nitrogen gas and 60-80 psiin case of in-line air) was regularly checked as was gas flow rate,seals, water levels in the cylinder filled with water and in the tank,and working temperatures.

[0258] After the pretreatment time elapsed, the reactors were moved outof the system and cooled down to ambient temperature. Samples were thenremoved for various analyses.

[0259] Biomass Washing Procedure

[0260] Washing and measurement procedures and mass calculations foruntreated and treated biomass were performed in a substantially similarmanner to that in Example 2.

[0261] Enzyme Hydrolysis

[0262] Lime-pretreated and washed biomass was transferred from thereactors to tubes with distilled water. Citrate buffer (1.0 M, pH 4.8)and sodium azide solution (1 (w/w) %) were added to the slurry to keepconstant pH and prevent microbial growth, respectively. Glacial aceticacid or saturated sodium hydroxide solution was then added to adjust thepH to approximately 4.8. The total volume of mixture was then increasedto the desired volume by adding distilled water. The tube was placed ina rotary shaker at 100 rpm and 50° C. After 1-hour incubation, cellulase(NREL, USA) and cellobiase (Novozyme 188, activity=250 CBU/g) were addedto the test tube. The loading rate of cellulase was 0, 1, 5, 10, 20, or60 FPU/g dry biomass and that of cellobiase was 28.5 CBU/g dry biomass.Samples were withdrawn at 0, 1, and 72 hours and sugars were measured ateach time point. The same procedure was also applied to untreatedbiomass.

[0263] Sugar Measurement

[0264] Reducing sugar was measured using the dinitrosalicylic acid (DNS)assay (Miller, 1959). A glucose standard prepared from the Sigma 100mg/dL glucose standard solution was used for the calibration, thus thereducing sugars were measured as “equivalent glucose”.

Example 4 Treatment of Corn Stover

[0265] Because raw corn stover has a broad particle size distribution,the particle size distributions in the two batches of corn stover usedin this example were compared to identify any batch to batch variation.

[0266] To compare particle sizes in the corn stover, the batches weresieved with USA standard testing sieves, which are well known in theart.

[0267] During the sieving, about 3.0 (w/w) % dry weight of corn stoverwas lost. The portion of large size particle, Tyler Mesh No. 28-4, ofthe second batch corn stover was about 4.0 (w/w) % smaller than that ofthe first batch corn stover (See Table 2). TABLE 2 The particle sizedistribution of the first and second batches of corn stover Range ofTyler Weight Contents (w/w) % Mesh Size First Batch Second BatchDifference* <100 3.75 4.49 0.73 100˜80 1.35 1.80 0.45  80˜48 5.68 6.841.16  48˜35 6.95 7.95 0.99  35˜28 8.68 9.44 0.77  28˜20 12.0 11.4 −0.64 20˜4 61.6 58.1 3.47

[0268] The major portion of particles (>60 (w/w) %) was large sizeparticles (Tyler Mesh No. 20-4). However, the particle size distributionfor two different batches was not significantly different (See FIG. 49).

[0269] The composition of the corn stover was analyzed by using it as alignocellulosic substrate. Its major components were cellulose,hemicellulose, lignin, and ash. In this experiment, the corn stovercompositions in the first and second batches were analyzed and thevariations between two batches were identified.

[0270] Untreated, washed corn stover was analyzed for moisture contentusing NREL Standard Procedure No. 001. Klason lignin content and acidsoluble lignin content were analyzed by NREL Standard Procedures No. 003and 004, respectively. Ash content was obtained by NREL StandardProcedure No. 005. Protein and mineral contents were determined byDepartment of Soil and Forage, Texas A&M University using standardprotocols.

[0271] The amounts cellulose and hemicellulose were estimated bysubtracting the above contents from 100%.

[0272] Lignin (Klason+acid-soluble lignin), protein, and other minorcontents were identical in both batches of corn stover. However, the ashcontent of the second batch corn stover was 2.45% lower than that of thefirst batch corn stover (See Table 3).

[0273] The lignin content of untreated, washed corn stover was notaffected by washing because almost same lignin content was found in rawcorn stover (20.9%). But the ash content of raw corn stover decreasedfrom 11.1% to 6.89% after washing alone. TABLE 3 Composition analysis ofuntreated, washed corn stover in batches one and two Lignin (%)Holocellulose* Acid- Batch No. (%) Klason soluble Total Ash (%) Protein(%) Others** (%) 1 70.4 18.5 2.49 21.0 6.89 0.78 0.95 2 73.6 17.8 2.4320.3 4.44 0.71 0.98 2-1*** 3.21 −0.65 −0.06 −0.71 −2.45 −0.07 0.03

[0274] The two batches of corn stover were pretreated with lime for 16weeks at 25, 35, 45, and 55° C. Both non-oxidative and oxidativeconditions were employed. The loading rates of lime and distilled waterwere 0.5 g Ca(OH)₂/g dry biomass and 10 mL water/g dry biomass,respectively. The lime-treated corn stover was harvested from eachreactor at 0, 1, 2, 4, 8, and 16 weeks.

[0275] Under the non-oxidative lime treatment conditions, less than 0.1g Ca(OH)₂/g dry biomass was consumed during 16 weeks. Lime consumptiondid not depend on temperature. After 16 weeks, the total protein contentdecreased from 0.78% in the untreated corn stover to 0.30% in thenon-oxidatively treated corn stover and 0.23% in the oxidatively treatedcorn stover at 55° C. On the other hand, in the oxidative limetreatment, much more than 0.1 g Ca(OH)₂/g dry biomass was consumed. Limeconsumption depended on temperature and thus the maximum amounts of limeconsumed oxidatively were 0.11, 0.14, 0.28, and 0.42 g Ca(OH)₂/g drybiomass at 25, 35, 45, and 55° C., respectively.

[0276] Temperature and oxygen without lime addition did notsignificantly affect the delignification of corn stover. Highertemperature in oxidative conditions with lime provided the highestamounts of delignification. This oxidative delignification followedfirst-order reaction kinetics.

[0277] Untreated and treated corn stover were hydrolyzed by cellulaseand cellobiase. The loading rate of cellulase was 0, 1, 5, 10, 20, and60 FPU/g dry biomass and that of cellobiase was 28.5 CBU/g dry biomass.The 3-day enzyme digestibility of the biomass increased dramaticallyduring the first few weeks in lime pretreatment. Oxidative limepretreatment rendered the corn stover more digestible than thenon-oxidative lime pretreatment. For instance, at a low cellulaseloading of 1 FPU/g dry biomass, the 3-day enzyme digestibility ofoxidatively treated corn stover was improved more than 77-109 mgequivalent glucose/g dry biomass compared with the non-oxidativetreatment for 16 weeks.

[0278] Four sets of packed bed PVC columns (D×L=1 inch×17 inch) wereconstructed for lime-pretreatment reactions at 25 (room temperature),35, 45, and 55° C. Each set was composed of two subsets, one with andone without aeration. The total number of columns for each subset was 10in order to be analyzed at five different run-times. Two columns wereharvested simultaneously at each run-time: 0, 1, 2, 4, 8, and 16 weeks.The treated biomass harvested from one of two columns was used toanalyze mass balance, lime consumption, lignin, protein and minerals,crystallinity, and acetyl group. The treated biomass from the othercolumn was dedicated to enzyme hydrolysis studies. Three sets of columnswith water jackets were operated at three different temperatures, 35,45, and 55° C., using the water heating and circulating system.

[0279] The water heating and circulating system included two parts:temperature controller and water circulator. The temperature controllercontained a temperature controller ({fraction (1/16)} DIN, OMEGA), athermocouple (KTSS-18G-18, OMEGA), a heating element (1.5 kW, 120 V), asolid-state relay (RSSDN-25A, Idec Co.), fuses (12.5 A and 0.25 A), anda main switch. The water circulator included a centrifugal pump (¾ HP,TEEL), a water tank (8 gal, Nalgene), a manifold having one input and 20output fittings, and return pipelines.

[0280] Air supplied by the Carter-Mattil compressor was preheated andsaturated in the cylinder within the water tank and then distributed toeach column by the air-manifold having one input and 10 output fittings.Compressed nitrogen gas (Plaxair Co.) was used for the non-oxidationcondition and supplied to each column by the N₂-manifold afterpreheating and saturation.

[0281]FIG. 47 is a schematic diagram of one subset of the jacketedreactor system for non-oxidative lime pretreatment. FIG. 48 shows theapparatus for oxidative lime pretreatment.

[0282] The solid content of the initial dried corn stover (iDCS) wasdetermined as described in NREL Standard Procedure No. 001. Corn stoverwas treated with lime, Ca(OH)₂, within each column. Each column wasdisassembled according to the time schedule and the analyticalexperiments were performed on the pretreated biomass.

[0283] Some small portions of biomass were retained inside of columnreactor when the column was disassembled to harvest the treated or theuntreated wet biomasses. Mass recovery yield was determined for thisstep and considered in the mass balance.

[0284] In order to examine mass recovery, after 1-hour incubation atambient temperature, reactors were disassembled. The wet biomass andlime mixture was harvested carefully from each reactor to 1-L centrifugebottle using sufficient amounts of distilled water. Without washing,lime concentration was directly determined by a neutralizing titrationmethod with 5-N HCl in a manner similar to that in Example 2. Thetitrated biomass was then centrifuged at 4,000 rpm for 15 minutes.Biomass slurry was obtained on the pre-weighed filter paper afterfiltration using aspirator. The solid content of the final dried cornstover (fDCS) was determined as described in NREL Standard Procedure No.001.

[0285] Mass recovery yield was 95.59±1.92% as shown in Table 4.

[0286] Lime recovery yield was 94.43±0.62%. TABLE 4 Mass recovery yieldafter column disassembly Trial Raw (g) Solid (%) iDCS (g) fDCS (g)Recovery 1 15.66 95.70 14.99 14.24 95.04% 2 15.66 95.70 14.99 14.0994.00% 3 15.66 95.70 14.99 14.65 97.73% Mean 95.59% STDEV 1.92%

[0287] Mass balance was determined to get the basic database in thisstudy of lime pretreatment of corn stover Mass recovery yields werelisted in Table 5. TABLE 5 Mass balance of treated corn stover innon-oxidative and oxidative conditions Non-oxidative ConditionsOxidative Conditions Time iDCS fDCS iDCS fDCS Temp. (weeks) (g) (g)Recovery (g) (g) Recovery 25° C. 1 15.14 12.96 85.63% 15.14 12.45 82.26%2 15.14 12.62 83.35% 15.14 12.29 81.16% 4 15.14 12.45 82.21% 15.14 12.0479.52% 8 15.14 12.22 80.74% 15.14 11.77 77.77% 15 15.06 11.61 77.09%15.06 10.73 71.23% 35° C. 1 15.02 12.50 83.21% 15.02 12.19 81.15% 215.12 12.14 80.27% 15.12 11.44 75.66% 4 15.12 11.99 79.28% 15.12 11.7177.44% 8 14.96 11.64 77.80% 14.96 11.40 76.22% 12 14.96 11.62 77.68%14.96 11.06 73.92% 16 14.96 11.73 78.38% 14.96 11.35 75.85% 45° C. 115.02 12.17 81.04% 15.02 11.79 78.46% 2 14.86 11.97 80.60% 14.86 11.4877.29% 4 14.86 11.72 78.87% 14.86 11.38 76.62% 8 14.93 11.54 77.30%14.93 11.55 77.33% 12 14.93 12.00 80.34% 14.93 11.13 74.53% 16 14.9311.56 77.43% 14.93 11.59 77.63% 55° C. 1 15.09 11.74 77.83% 15.09 11.5376.40% 2 15.05 11.55 76.70% 15.05 11.37 75.53% 4 15.09 11.56 76.63%15.09 12.48 82.69% 6 15.09 11.45 75.92% 15.09 11.59 76.79% 8 15.05 11.2374.63% 15.05 12.54 83.29% 12 15.05 11.06 73.45% 15.05 11.57 76.84% 1614.97 11.44 76.39% 14.97 11.48 76.69%

[0288] The amount of lime consumed during the pretreatment wasdetermined by titrating with 5-N HCl solution at pH 7.0. Certified 5-NHCl was used to determine the remaining amounts of lime in the treatedbiomass mixture. The lime-treated biomass was harvested from the columnreactor and transferred into a 1-L centrifuge bottle. 5-N HCl wasgradually added to neutralize the treated biomass mixture until pH 7.0.During the titration, the pH of the mixture was measured while agitatingcontinuously. The amount of 5-N HCl used for titration was recorded toestimate the amount of lime unreacted in the mixture (R) using thefollowing formula:${R(g)} = \frac{{Mw} \times \Delta \quad V \times N}{2 \times 1000}$

[0289] where Mw=molecular weight of lime.

[0290] ΔV=volume of 5-N HCl titrated,and

[0291] N=normality concentration of HCl.

[0292] The amount of lime consumed (C) during the pretreatment wasestimated from the following mass balance for lime: C (g)=the initialamount of lime in reactor−R.

[0293] During the non-oxidative lime treatment, less than 0.1 gCa(OH)₂/g dry biomass was consumed during 16 weeks. The maximum amountof lime consumed was 0.07 g Ca(OH)₂/g dry biomass. Lime consumption didnot depend on temperature in non-oxidative pretreatment conditions (FIG.50). Under oxidative lime pretreatment conditions, however, the amountof lime consumed did depend on temperature. Lime consumption increasedas temperature increased (FIG. 51). The maximum amounts of lime consumedoxidatively were 0.11, 0.14, 0.28, and 0.42 g Ca(OH)₂/g dry biomass at25, 35, 45, and 55° C., respectively.

[0294] As shown above, the lignin content of corn stover was notaffected with washing only. Additional experiments similar to thoseabove also showed that lignin content was not substantially affectedabsent addition of lime. Non-oxidative treatment without lime wasstudied to identify the temperature effect on delignification. Oxidativetreatment without lime was studied to identify the combined effect oftemperature and aeration on delignification. Oxidative researchconditions were achieved by aerating at 25 and 55° C.

[0295] 15.0 g of corn stover and 150.0 mL of distilled water were loadedin column reactors, which were operated as the same procedure describedabove for pretreatment, except that no lime was added.

[0296] Non-oxidative and oxidative conditions without lime were achievedby purging nitrogen gas and air during the 10-week operation at 25 and55° C., respectively.

[0297] The treated corn stover was used to determine Klason,acid-soluble, and total lignin contents. Analytical methods weredescribed in NREL Standard Procedures No. 003 and 004.

[0298] There were no significant effects of temperature or aeration ondelignification as shown in Table 6. TABLE 6 Comparison of lignincontents of untreated corn stover both non-oxidative and oxidativeconditions without lime addition* Lignin Content Temperature Acid-Condition (° C.) Klason (%) soluble (%) Total (%) Non- 25 19.34 2.0021.34 oxidative 55 19.90 1.64 21.54 Oxidative 25 19.27 2.01 21.28 5518.72 1.55 20.27 Control** — 18.50 2.49 21.00

[0299] Delignification of corn stover was achieved by lime treatment.Non-oxidative treatment with lime was used to identify the temperatureeffect on delignification. Oxidative treatment with lime was used toidentify the combined effect of temperature and aeration ondelignification.

[0300] Corn stover was treated with lime in non-oxdiative and oxidativeconditions. The treated corn stover was used to determine Klason,acid-soluble, and total lignin contents. Analytical methods weredescribed in NREL Standard Procedures No. 003 and 004.

[0301] After non-oxidative lime pretreatment, Klason lignin contentdecreased from 19.6% down to 13%. Delignification occurred significantlywithin the first 2 weeks of treatment but did not depend on temperatureafter around 4 weeks (FIG. 52).

[0302] In contrast, during oxidative pretreatment, the Klason lignincontent decreased significantly throughout the entire treatment time.Delignification depended on temperature at this condition (FIG. 53).

[0303] During the non-oxidative lime pretreatment, acid-soluble lignincontent decreased from 1.8% to 1.2%. The reduction tendency ofacid-soluble lignin was similar to that of Klason lignin (FIG. 54).

[0304] Under oxidative pretreatment, however, acid-soluble lignincontents started to decrease for the first 2 weeks, but graduallyrecovered after 2 weeks, even though the increase was relatively smallcompared with Klason lignin contents. The recovering rate ofacid-soluble lignin also increased as temperature increased as shown inFIG. 55.

[0305] During the 16-week lime pretreatment, non-oxidativedelignification removed up to 29.1, 32.9, 29.2, and 31.8% of lignin at25, 35, 45, and 55° C., respectively. Oxidative delignification,however, removed up to 40.9, 48.0, 61.8, and 67.7% of lignin at 25, 35,45, and 55° C., respectively during the same period.

[0306] Delignification by oxidative lime pretreatment followedfirst-order kinetics expressed as following rate equation:$\frac{L}{t} = {k \cdot L}$

[0307] where L=total lignin content (=Klason lignin+acid solublelignin), and

[0308] k=rate constant of delignification.

[0309] The integrated form of this equation is

1nL=−k·t+1nL ₀

[0310] where L₀=Initial total lignin content.

[0311] The result of regression analysis with SAS for data obtained inthis example is summarized in Table 7. Fitting results for the data ofnon-oxidative lime pretreatment were poor, but the data for oxidativetreatment fit the integrated equation very well.

[0312] The delignification rate constant (k) is a function oftemperature, thus it can be expressed in the Arrhenius equation asfollows:

k=k _(o) exp(−E _(a) /RT)

[0313] where k_(o)=pre-exponential factor (1/week),

[0314] E_(a)=activation energy (Joule/mol),

[0315] R=ideal gas constant, 8.314 Joule/(mol·K),

[0316] T=absolute temperature (K),

[0317] The Arrhenius plot is shown in FIG. 56.

[0318] From the data listed in Table 7 and FIG. 56, k_(o) and E_(a) weredetermined.

[0319] Activation energy (E_(a)) for oxidative delignification wasdetermined as follows:

Slope=−E _(a) /R=−2973.5 K,

[0320] thus E_(a)=(2973.5)×(8.314)=24.72 kJ/mol. TABLE 7 Results oflinear regression analysis for delignification data of limepretreatement. lnL₀ L₀ Condition (g lignin/ (g lignin/ Regression forLime Temp. g dry g dry k Coefficient Pretreatment (° C.) biomass)biomass) (week⁻¹) (R² ) Non- 25 −1.7336 0.1767 0.0099 0.7919 oxidative35 −1.8177 0.1624 0.0075 0.8830 45 −1.8259 0.1611 0.0077 0.4484 55−1.8720 0.1538 0.0032 0.5595 Oxidative 25 −1.7421 0.1752 0.0214 0.951635 −1.8380 0.1591 0.0270 0.9225 45 −1.8668 0.1546 0.0460 0.9661 55−1.9959 0.1359 0.0483 0.9026

[0321] Lime treatment increased the holocellulose content due to thereduction of lignin content (FIG. 57).

[0322] To compare their digestibilities, untreated and treated cornstovers were hydrolyzed to monosaccharides by cellulase and cellobiase.The digestibilities of corn stover treated with non-oxidative andoxidative lime at 25, 35, 45, and 55° C. were also determined.

[0323] Substrates were the untreated, washed, the non-oxidativelytreated, and the oxidatively treated corn stovers. Enzyme reactionprocedures were standard procedures described in Example 3.

[0324] The 3-day enzyme digestibility of untreated corn stover was 153and 193 mg equiv. glucose/g dry biomass at 5 and 60 FPU/g dry biomass ofenzyme (cellulase) loading, respectively. Enzyme hydrolysis profiles(FIG. 58) fit well to the following equation:

Y=A·ln(X)+B

[0325] where Y=sugar yield (mg equivalent glucose/g dry biomass),

[0326] X=cellulase loading rate (FPU/g dry biomass), and

[0327] A and B are empirical constants.

[0328] During the 16-week non-oxidative lime pretreatment, 3-day enzymedigestibility increased 3-fold more than of the untreated corn stoverover the entire range of cellulase concentrations (FIG. 59).

[0329] Under most conditions, 3-day enzyme digestibility increaseddramatically for the first few weeks and increased continuously for theremaining treatment. Interestingly, the 3-day enzyme digestibility ofnon-oxidatively treated corn stover at 55° C. reached the maximum aftera 4-week lime pretreatment (FIG. 60).

[0330] During the 16-week oxidative lime pretreatment, the 3-day enzymedigestibility increased by more 15-123 mg equivalent glucose/g drybiomass than that of the 16-week non-oxidative lime pretreatment (FIG.63 and Table 6.1). The improvement of 3-day enzyme digestibility fromnon-oxidative values to oxidative values depended on the cellulaseloading: the lower the cellulase loading, the greater improvement of3-day enzyme digestibility. The 3-day enzyme digestibility profiles ofthe 16-week oxidatively treated corn stover were similar to those of thenon-oxidatively treated corn stover (FIG. 61).

[0331] In contrast, oxidative lime treatment shortened the pretreatmenttime required to obtain maximal 3-day enzyme digestibility at highertreatment temperatures (See FIG. 62 and Table 6.2).

[0332] For example, using a cellulase loading only of 1 FPU/g drybiomass, the 3-day enzyme digestibility of the oxidatively treated cornstover improved more than 77-109 mg equivalent glucose/g dry biomasscompared with the non-oxidative treatment for 16 weeks (FIG. 63).

[0333] It is likely that enhanced 3-day enzyme digestibility mainlyresults from lime reaction, which is boosted by the presence of oxygen.Higher temperatures are more favorable because they result in greaterdeliginification, which results in the faster digestion of biomass.TABLE 8 Differences* in 3-day enzyme digestibility between non-oxidativeand oxidative treated corn stover treated for 16 weeks Cellulose Loading(FPU/g dry biomass) Temp. (° C.) 1 5 10 20 60 25 77.24 123.10 31.5451.88 44.65 35 67.18 44.26 55.44 83.13 32.54 45 121.71 46.40 64.83 46.4315.35 55 109.10 42.75 109.64 87.23 57.93

[0334] TABLE 9 The minimal oxidative treatment time (t500) required toobtain greater than 500 mg equivalent glucose/g dry biomass of 3-denzyme digestibility at 1 FPU/g dry biomass of cellulase loading (Basedon the data of FIG. 63) Temperature (° C.) t₅₀₀ (weeks) 25 >16 35 16 458 55 4

[0335] Total protein content of the oxidatively treated corn stover wasmuch lower than that of the non-oxidatively treated corn stover as shownin FIG. 64

1. A system for processing biomass, comprising: a water-impermeablebottom liner; a gravel layer supported by the bottom liner; a drain pipedisposed within the gravel layer; a biomass input device operable todeliver biomass over the gravel layer to form a biomass pile; a limeinput device operable to deliver lime to the biomass for pretreating thebiomass; a distribution pipe elevated above the gravel layer; and a pumpoperable to circulate water through the biomass pile by delivering waterto the distribution pipe and receiving water from the drain pipe afterit has traveled through the biomass pile.
 2. The system of claim 1,wherein the biomass is lignocellulosic biomass.
 3. The system of claim1, wherein the lignocellulosic biomass is selected from the groupconsisting of bagasse and corn stover.
 4. The system of claim 1, whereinthe gravel layer is approximately three feet thick.
 5. The system ofclaim 1, wherein the lime input device is operable to deliver lime tothe biomass either during or after the delivering of the biomass overthe gravel layer.
 6. The system of claim 1, wherein the lime inputdevice is operable to deliver lime to the biomass in an amount betweenapproximately 10% and 30% of the biomass by weight.
 7. The system ofclaim 1, further comprising an inoculum input device operable to deliveran inoculum to the biomass pile for fermentation of the biomass pile. 8.The system of claim 1, further comprising a heat exchanger coupled tothe distribution pipe and operable to control a temperature of the waterthat is delivered to the distribution pipe.
 9. The system of claim 1,further comprising an air blower and an air distribution pipe operableto deliver air to the biomass pile.
 10. The system of claim 9, furthercomprising a container of lime water slurry coupled to the airdistribution pipe and operable to scrub the air of carbon dioxide beforethe air is delivered to the biomass pile.
 11. The system of claim 1,further comprising a calcium carbonate input device operable to delivercalcium carbonate to the biomass for pretreating the biomass.
 12. Asystem for processing biomass, comprising: a water-impermeable bottomliner; a grid-like lattice structure coupled to the bottom liner to forma roof; a geomembrane coupled to the grid-like lattice structure; agravel layer supported by the bottom liner; a plurality of drain pipesdisposed within the gravel layer; a conveyor belt coupled to the topliner and operable to deliver biomass over the gravel layer to form abiomass pile; a lime input device operable to deliver lime to thebiomass for pretreating the biomass; a plurality of distribution pipescoupled to the top liner and associated with respective ones of theplurality of drain pipes; and a plurality of pumps coupled to respectiveones of the plurality of drain pipes and respective ones of theplurality of distribution pipes, the pumps operable to circulate waterthrough the biomass pile by delivering water to the distribution pipesand receiving water from the drain pipes after the water has traveledthrough the biomass pile.
 13. The system of claim 12, wherein thebiomass is lignocellulosic biomass selected from the group consisting ofbagasse and corn stover.
 14. The system of claim 12, wherein thegrid-like lattice structure is formed from a plurality of I-beams in ageneral shape of a half cylinder.
 15. The system of claim 12, furthercomprising a foam layer coupled to an outside of the geomembrane. 16.The system of claim 12, further comprising a sugar extraction deviceoperable to extract sugar from a raw feedstock to produce the biomass.17. The system of claim 16, wherein the raw feedstock is selected fromthe group consisting of energy cane and sweet sorghum.
 18. The system ofclaim 16, wherein the sugar extraction device comprises a plurality ofadjacent extraction tanks, each extraction tank comprising: a screwconveyor operable to deliver solid material from the raw feedstock an adownstream direction; and a weir operable to deliver liquid materialfrom the raw feedstock in an upstream direction.
 19. The system of claim12, wherein the lime input device is operable to deliver lime to thebiomass either during or after the delivering of the biomass over thegravel layer.
 20. The system of claim 12, further comprising an inoculuminput device operable to deliver an inoculum to the biomass pile forfermentation of the biomass pile.
 21. The system of claim 12, furthercomprising a heat exchanger coupled to the distribution pipe andoperable to control a temperature of the water that is delivered to thedistribution pipe.
 22. The system of claim 12, further comprising an airblower and an air distribution pipe operable to deliver air to thebiomass pile.
 23. The system of claim 22, further comprising a containerof lime water slurry coupled to the air distribution pipe and operableto scrub the air of carbon dioxide before the air is delivered to thebiomass pile.
 24. The system of claim 12, further comprising a calciumcarbonate input device operable to deliver calcium carbonate to thebiomass for pretreating the biomass.
 25. A system for processingbiomass, comprising: an end wall; a water-impermeable bottom liner; atop liner coupled to the bottom liner, the top liner selectivelyinflatable by one or more fans coupled to the end wall; a plurality ofwater pouches coupled to the top liner, the water pouches selectivelyinflatable when the top liner is inflated; a gravel layer supported bybottom liner and separated into a plurality of gravel segments; aplurality of drain pipes disposed within respective ones of the gravelsegments; a conveyor belt associated with the end wall and operable todeliver biomass over the gravel segments to form a biomass pile; a limeinput device operable to deliver lime to the biomass for pretreating thebiomass; a plurality of distribution pipes coupled to the top liner andassociated with respective ones of the plurality of gravel segments; anda plurality of pumps coupled to respective ones of the plurality ofdrain pipes and respective ones of the plurality of distribution pipes,the pumps operable to circulate water through the biomass pile bydelivering water to the distribution pipes and receiving water from thedrain pipes after the water has traveled through the biomass pile. 26.The system of claim 25, wherein the biomass is lignocellulosic biomassselected from the group consisting of bagasse and corn stover.
 27. Thesystem of claim 25, further comprising an opening formed in the end wallfor unloading residue left over from the biomass pile afterfermentation.
 28. The system of claim 25, further comprising a sugarextraction device operable to extract sugar from a raw feedstock toproduce the biomass.
 29. The system of claim 28, wherein the rawfeedstock is selected from the group consisting of energy cane and sweetsorghum.
 30. The system of claim 28, wherein the sugar extraction devicecomprises a plurality of adjacent extraction tanks, each extraction tankcomprising: a screw conveyor operable to deliver solid material from theraw feedstock an a downstream direction; and a weir operable to deliverliquid material from the raw feedstock in an upstream direction.
 31. Thesystem of claim 25, wherein the lime input device is operable to deliverlime to the biomass either during or after the delivering of the biomassover the gravel layer.
 32. The system of claim 25, further comprising aninoculum input device operable to deliver an inoculum to the biomasspile for fermentation of the biomass pile.
 33. The system of claim 25,further comprising a heat exchanger coupled to the distribution pipe andoperable to control a temperature of the water that is delivered to thedistribution pipe.
 34. The system of claim 25, further comprising an airblower and an air distribution pipe operable to deliver air to thebiomass pile.
 35. The system of claim 34, further comprising a containerof lime water slurry coupled to the air distribution pipe and operableto scrub the air of carbon dioxide before the air is delivered to thebiomass pile.
 36. The system of claim 25, further comprising a calciumcarbonate input device operable to deliver calcium carbonate to thebiomass for pretreating the biomass.
 37. A system for processingbiomass, comprising: a plurality of geodesic domes arranged in agenerally circular pattern, each geodesic dome comprising: awater-impermeable bottom liner; a top liner coupled to the bottom liner;a gravel layer supported by the bottom liner; a drain pipe disposedwithin the gravel layer; and a distribution pipe elevated above thegravel layer; a plurality of pumps coupled to respective ones of theplurality of geodesic domes, each pump operable to circulate waterthrough its respective geodesic dome by delivering water to thedistribution pipe associated with the respective geodesic dome andreceiving water from the drain pipe associated with the respectivegeodesic dome; a rotatable conveyor belt surrounded by the geodesicdomes and operable to deliver biomass to each geodesic dome; and a limeinput device operable to deliver lime to the biomass for pretreating thebiomass.
 38. The system of claim 37, wherein the biomass islignocellulosic biomass selected from the group consisting of bagasseand corn stover.
 39. The system of claim 37, wherein each top linercomprises a plurality of hexagonal or pentagonal panels coupled to oneanother with lips associated with each panel.
 40. The system of claim37, further comprising a foam layer coupled to an outside of the topliner.
 41. The system of claim 37, wherein the lime input device isoperable to deliver lime to the biomass either during or after thedelivering of the biomass over the gravel layer.
 42. The system of claim37, further comprising a calcium carbonate input device operable todeliver calcium carbonate to the biomass for pretreating the biomass.43. A system for processing biomass, comprising: a fermenter structureconfigured to: accept and store untreated lignocellulosic biomass;pretreat the lignocellulosic biomass with lime at a temperature betweenapproximately 25° C. and 95° C. at ambient pressure for a time period ofat least approximately four weeks; and treat the lignocellulosic biomasswith an inoculant.
 44. A method of biomass pretreatment comprising:adding an alkali to biomass with lignin content to produce a mixture;and incubating the mixture at a temperature between approximately 25° C.and 95° C. at ambient pressure.
 45. The method of claim 44, furthercomprising incubating the mixture for a time period of at leastapproximately 4 weeks.
 46. The method of claim 44, further comprisingincubating the mixture for a time period of between approximately 4 and16 weeks.
 47. The method of claim 44, further comprising selecting theduration of incubation based on incubation temperature.
 48. The methodof claim 44, wherein the biomass comprises lignocellulosic biomass. 49.The method of claim 44, wherein the biomass comprises agriculturalwaste.
 50. The method of claim 44, wherein the biomass is selected fromthe group consisting of: bagasse, corn stover and combinations thereof.51. The method of claim 44, further comprising circulating water throughthe biomass during incubation.
 52. The method of claim 44, furthercomprising circulating air through the biomass during incubation. 53.The method of claim 44, further comprising circulating oxygen enrichedair through the biomass during incubation.
 54. The method of claim 44,wherein the alkali comprises lime.
 55. The method of claim 44, whereinthe alkali comprises calcium oxide.
 56. The method of claim 54, furthercomprising adding approximately 0.5 grams of lime per gram of biomass toproduce the mixture.
 57. The method of claim 54, further comprisingadding approximately 0.1 to 0.5 grams of lime per gram of biomass toproduce the mixture.
 58. The method of claim 54, further comprisingadding lime to the biomass in an amount between approximately 10% and30% of biomass by weight.
 59. The method of claim 44, further comprisingadding calcium carbonate to the mixture.
 60. The method of claim 44,further comprising incubating the mixture at a temperature betweenapproximately 25° C. and 90° C.
 61. The method of claim 44, furthercomprising incubating the mixture at a temperature between approximately25° C. and 57° C.
 62. The method of claim 44, further comprisingselecting the incubation temperature based on the partial pressure ofwater at the selected temperature.
 63. The method of claim 44, furthercomprising increasing the enzyme digestibility of the biomass.
 64. Themethod of claim 44, further comprising producing pulp.
 65. The method ofclaim 64, further comprising producing pulp suitable for paper orcardboard production.
 66. The method of claim 44, further comprisingreducing the lignin content of the biomass.
 67. The method of claim 66,further comprising reducing lignin content by at least approximately98%.
 68. The method of claim 66, further comprising reducing lignincontent by at least approximately 90%.
 69. The method of claim 66,further comprising reducing lignin content by at least approximately29%.
 70. The method of claim 66, further comprising reducing lignincontent by at least approximately 40%.
 71. The method of claim 66,further comprising reducing lignin content by at least approximately67%.
 72. The method of claim 66, further comprising reducing lignincontent by alkaline oxidation.
 73. The method of claim 44, furthercomprising fermenting the biomass after incubation.
 74. The method ofclaim 73, further comprising adding an inoculum to the mixture afterincubation.
 75. The method of claim 73, further comprising collectingcarboxylate salts from the mixture.
 76. The method of claim 73, furthercomprising placing the mixture prior to incubation in a storage facilitysuitable for incubation and fermentation.
 77. A method for producingenzymatically digestible biomass comprising: adding lime to biomass withlignin content to produce a mixture; incubating the mixture at atemperature between approximately 25° C. and 55° C. at ambient pressurefor a time period of at least approximately 4 to 16 weeks; circulatingwater through the mixture during incubation.
 78. The method of claim 72,further comprising circulating air through the mixture duringincubation.
 79. The method of claim 77, further comprising reducing thelignin content of the biomass by at least approximately 67%.
 80. Themethod of claim 22, further comprising reducing the lignin content ofthe biomass by at least approximately 32%.
 81. The method of claim 22,further comprising fermenting the biomass after incubation.
 82. A methodfor producing pulp comprising: adding lime to biomass with lignincontent to produce a mixture; incubating the mixture at a temperaturebetween approximately 45° C. and 55° C. at ambient pressure for a timeperiod of approximately 10 weeks; circulating water through the mixtureduring incubation.
 83. The method of claim 82, further comprisingcirculating air through the mixture during incubation.
 84. The method ofclaim 82, further comprising reducing the lignin content of the biomassby at least approximately 90%.
 85. The method of claim 82, furthercomprising reducing the lignin content of the biomass by at leastapproximately 40%.
 86. The method of claim 82, further comprisingproducing paper or cardboard from the biomass after incubation.